Disposable cartridge for characterizing particles suspended in a liquid

ABSTRACT

The present invention relates to a disposable cartridge for characterizing particles suspended in a liquid, especially a self-contained disposable cartridge for single-use analysis, such as for single-use analysis of a small quantity of whole blood. The self-contained disposable cartridge facilitates a straightforward testing procedure, which can be performed by most people without any particular education. Furthermore, the apparatus used to perform the test on the cartridge is simple, maintenance free, and portable.

The present invention relates to a disposable cartridge forcharacterizing particles suspended in a liquid, especially aself-contained disposable cartridge for single-use analysis, such as forsingle-use analysis of a small quantity of whole blood. Theself-contained disposable cartridge facilitates a straightforwardtesting procedure, which can be performed by most people without anyparticular education. Furthermore, the apparatus used to perform thetest on the cartridge, could be made simple, light and maintenance free,thus giving full portability and a large range of operation for theuser. The invention provides steps for pre-analytic handling of samplessuch as hemolysing of red blood cells and inactivation of coagulation.

Present instruments for particle characterization such as counting andsizing are fairly expensive, immobile and require operation by trainedpersonnel. The consequence hereof has been that many instruments areplaced in dedicated laboratories that are operated by specializedpersonnel. Furthermore, the samples to be analysed must be transportedto this laboratory and the results are reported back to the requiree.

In WO 01/11338, which is hereby incorporated by reference, an apparatusis disclosed for characterizing particles suspended in a liquid,comprising a disposable cartridge and a docking station for removablyreceiving the cartridge. The cartridge comprises a housing with a firstcollection chamber bounded by a wall containing an orifice for thepassage of the particles and having an inlet/outlet for connection to asource of positive or negative gas pressure, and components of aparticle characterization device for characterizing particles passingthrough the orifice that are connectable from outside the housing. Thedocking station comprises a port for connection with a source ofpositive or negative gas pressure and forming a gas connection with theinlet/outlet when the cartridge is received in the docking station, andmeans for operative connection with the components of a particlecharacterization device when the cartridge is received in the dockingstation.

In WO 02/089670, which is hereby incorporated by reference, a device forsampling a small and precise volume of liquid is disclosed, comprising amovable member with a cavity for entrapment and displacement of anaccurate part of a liquid sample.

It is a disadvantage of these prior art devices that several devices areused to perform an analysis, e.g. of a whole blood sample. The sampletaking is performed with a separate device, and the sample has to betransferred to another device for sample preparation before it isfinally transferred to a sensor for analysis.

In WO 99/01742 a disposable sampling device is disclosed for anapparatus for counting particles contained in a liquid. The samplingdevice is connectable in a defined position to the apparatus. The devicehas means for introducing a sample therein, means for metering a definedvolume of the sample, means containing a defined volume of a dilutingliquid, a diluting chamber, means for simultaneously directing thedefined volume of sample and the defined volume of diluting liquid tothe diluting chamber for obtaining therein a diluted sample, means fordirecting at least a portion of the diluted sample past particlecounting means and signal transmitting means connecting the particlecounting means and terminal means located at an outer boundary of thehousing in a position corresponding to a location of terminal means ofthe apparatus when the housing is connected thereto in the definedposition.

During blood analysis with the device described in WO 99/01742, theblood sample is pumped back and forth several times for dilution, mixingand analysis, and the flow system is closed so that the pressure in thesystem is increased and decreased above and below, respectively,atmospheric pressure during movement of the sample. Further, sampletaking requires pumping with a membrane or another flow actuator causingentrance of blood into the flow system of the device. Thus, the abovedisclosed flow system is rather complicated.

The particle counting is, as described in WO 99/01742, performed in anopen-ended tube so that the volume of diluted sample passing theparticle counting sensor is very small.

The blood analysis, as described in WO 99/01742 does not take intoaccount that particles of different kind and concentration might needpre-analytic separation, decomposition, staining or labeling in order tobe accurately recorded by the sensing principle in account.

The blood test sequence as described in WO 99/01742 does not take intoaccount that users without prior education herein should be able tolearn how to perform this test themselves, i.e. no pre-analyticaldilution steps should be required.

Thus, it is an object of the present invention to provide a cartridgefor characterizing particles suspended in a liquid that enables sampletaking, sample preparation, and particle characterization so thatanalysis may be performed within one device without a need for samplehandling and sample transfer to another unit.

It is a further object of the present invention to provide a cartridgethat is adapted for single-use to be discarded after analysis of oneliquid sample.

It is another object of the present invention to provide a cartridgethat has a simple flow system.

It is yet another object of the present invention to provide a flowsystem in the cartridge communicating with the surroundings so that thepressure in the flow system remains substantially constant atatmospheric pressure.

According to the present invention, the above-mentioned and otherobjects are fulfilled by a cartridge for characterizing particlessuspended in a liquid, comprising a housing with a first mixing chamberand a first collection chamber separated by a wall containing an orificefor passage of the particles between the first mixing chamber and thefirst collection chamber. Particle characterization means are providedfor characterizing particles passing through the orifice.

Sample taking may be performed through a bore in the outer surface ofthe housing for entrance of a liquid sample. The housing furthercomprises a sampling member that is movably positioned in the housing.The sampling member has a first cavity for receiving and holding a smalland precise volume of liquid. In a first position of the samplingmember, the first cavity is in communication with the bore for entranceof the liquid sample into the first cavity, and, in a second position ofthe sampling member, the first cavity is in communication with an inletto the first mixing chamber.

Thus, the sampling member operates to receive and hold a precise volumeof liquid sample and to transfer the sample to the inlet of the firstmixing chamber.

Preferably, liquid to be sampled enters the cavities by capillaryattraction causing a liquid flow. Utilization of capillary forcessimplify the flow system, since no pumps, membranes, syringes or otherflow generating means are, in contrast to WO 99/01742, needed to takethe sample.

Thus, the bore may form a first capillary tunnel for entrance of aliquid sample by capillary attraction. The capillary tunnel isdimensioned so that, upon contact between the bore and liquid to besampled, a sample of the liquid is drawn into the bore by capillaryattraction.

Further, the first cavity may form a second capillary tunnel adapted fordrawing the liquid sample into the first cavity by capillary attraction.Preferably, the first and second capillary tunnel has the same diameter,and it is also preferred that, in the first position, the first andsecond capillary tunnel extend along substantially the same longitudinalcenter axis.

Preferably, the sampling member is rotatable about an axis of rotationthat is substantially perpendicular to a longitudinal axis of the firstcavity.

Additionally or alternatively, the sampling member may be displaced in adirection substantially perpendicular to a longitudinal axis of thefirst cavity.

The surface of the first and second inner capillary tunnel walls may behydrophilic whereby the capillary attraction of the liquid sample isfacilitated. For example, the inner tunnel walls may be made of e.g.glass or polymers, such as polystyrene.

Alternatively, the capillary tunnel walls may be made of another type ofmaterial and covalently or non-covalently coated with a hydrophilicmaterial, such as a polymer or a reagent.

The capillary tunnel may also include one or more reagents adhered orchemically bonded to the inner tunnel wall. These reagents serve thepurposes of further facilitating the capillary attraction of the sampleand optionally also causing a chemical reaction in the liquid sample,e.g. introducing anticoagulant activity in a blood sample. Such reagentsmay comprise heparin, salts of EDTA, etc.

Preferably, the sampling member is made of a polymer.

In accordance with a further aspect of the invention, an apparatus isprovided for characterizing particles suspended in a liquid, comprisinga cartridge as disclosed herein, and a docking station for removablyreceiving the cartridge, the docking station comprising connectors foroperational connection with the particle characterization means when thecartridge is received in the docking station.

The cartridge may further comprise a cartridge port communicating withthe first collection chamber for causing a liquid flow through theorifice, and the docking station may further comprise a correspondingport for forming a gas connection with the cartridge port when thecartridge is received in the docking station for application of apressure causing a liquid flow through the orifice.

The particle characterization means may include a first electrode in thefirst mixing chamber and a second electrode in the first collectionchamber, each electrode being electrically connected to a respectiveterminal member accessible at the outer surface of the cartridge foroperational connection to the respective connector of the dockingstation when the cartridge is received in the docking station.Generally, it is preferred that all necessary electrical and fluidconnections to the cartridge can be established by fitting the cartridgeinto the docking station, preferably by a simple push fit.

The first and second electrodes may facilitate particle characterizationutilizing the well-known Coulter impedance principle, e.g. for countingand sizing of blood cells. This method has become a globally acceptedmethod and is being used in the majority of haematology-analysers.Several thousand particles per second may be characterized with highprecision and accuracy utilizing this principle.

With the electrical impedance technique it is possible to resolve theparticle volume from the measurement. By maintaining a constant currentacross the orifice, the recorded voltage pulse from particles displacingthe electrolyte in the orifice will have a height proportional to thevolume of the particle. This is because particles can be considerednon-conducting compared to the electrolyte, the electrical field (DC orRF) in the centre of the orifice is homogeneous, which is normally thecase when the diameter D is smaller than the length l of the orifice(I/D>1), the particle d is to be considered small compared to thediameter of the orifice (d<0.2*D), only one particle passes through at atime and the particles are passed through the orifice along the lengthof the orifice.

Normally such apparatus is operated so that the flow through the orificeis into the first collection chamber.

Preferably, the length of the orifice is from 1 to 1000 μm, for exampleabout 50 μm. Desirably the length of the orifice is chosen such thatonly one particle will be present in the orifice at the time whendetecting particles of from 0.1 to 100 μm diameter. However,considerations to the homogeneity of the electrical field in the orificemay require a length of the orifice larger or equal to the diameter. Thecounts, of which some may be simultaneous counting of two particles, canbe corrected mathematically by implementing a statistical estimation.The aspect ratio of the orifice, (length or depth divided by diameter)is preferably from 0.5:1 to 5:1, more preferably from 1:1 to 3:1.

Preferably, the largest cross-sectional dimension of the orifice is from5 to 200 μm, for example 10 to 50 μm.

As explained above, the present invention provides in preferred aspectsa sensor based on a membrane fabricated in e.g. a polymer sheet by laserablation. The membrane has an orifice placed relatively in the centre ofthe membrane, which can be used for aspiration of particles suspended ina liquid, as the sensor is submerged into the liquid. This way oftransporting particles into a measuring region is known for electricalcharacterization of particles by the Coulter principle (V. Kachel,“Electrical Resistance Pulse Sizing: Coulter Sizing”, Flow Cytometry andSorting, 2. ed., pp 80,1990 Wiley-Liss, Inc.).

The cartridge may further comprise a breather inlet/outlet communicatingwith the surroundings for preservation of substantially ambientatmospheric pressure in the cartridge flow system for facilitation ofliquid flow through the orifice.

Preferably, the cartridge is designed to be disposable after a singleuse. It is desirable that after use there is no need to clean theapparatus before it can be used in a new assay procedure with a newcartridge. Accordingly, escape of liquid from the cartridge at its entryinto the docking station should be avoided. To this end the positioningof the orifice with respect to the breather inlet/outlet, the secondchamber inlet/outlet and the particle characterization device componentsis preferably such that a volume of liquid sufficient for the desiredparticle characterization can be drawn or pumped through the orificewithout the liquid passing out of the housing. Generally, it should bepossible to pass a volume of liquid, which is at least 0.1 ml to 10 ml,e.g. 0.5 ml, through the orifice whilst particle characterizationmeasurements are being made with no liquid leaving the cartridge.

The cartridge may comprise volume-metering means for determining thebeginning and end of a period during which a predetermined volume ofliquid has passed through the orifice.

Preferably, the volume metering means comprises a volume-meteringchamber with an input communicating with the first collection chamberand an output, and wherein presence of liquid is detected at the inputand at the output, respectively.

For example, presence of liquid may be detected optically due to changedoptical properties of a channel configuration from being filled with airtill when it is being filled with liquid. This could be constructed asreflectance or transmittance detection from the surface, where incidentlight is reflected from an empty channel and transmitted through afilled channel, thus giving a clear shift in the detected reflected ortransmitted light.

It is preferred that the input and output of the metering chamber isformed by narrow channels for accommodation of only a small liquidvolume compared to the volume of the metering chamber so that the actualpositioning of the volume metering means, e.g. optical reflectancedetection, in the channels do not substantially influence the accuracyof the volume metering means determination.

The first mixing chamber or the first collection chamber may constitutethe volume metering chamber; however, it is preferred to provide anindependent volume metering chamber facilitating positioning of thevolume metering means, e.g. the optical reflectance detection.

The volume metering means may be positioned for sensing when liquid inthe metering chamber is at or above respective levels in thevolume-metering chamber.

The volume metering means may be used for sensing when the level of theliquid is such that the respective metering means are or are not filledwith the liquid and may therefore serve for determining the beginningand end of a period during which a fixed volume of liquid has passedthrough the orifice. For example, particle characterization may beginwhen the level of the liquid just rises over the level of a firstmetering means and may end when the level of the liquid just rises overa second metering means, the volume of liquid passing through theorifice during this period being defined by the separation of therespective metering means.

Where the end point of the passage of a defined volume of liquid is theeffective emptying of one chamber to below the level of the orifice, itis preferred that each of the collection and first mixing chambers (orat least that chamber from which liquid passes) has a transverse crosssectional area at the level of the orifice which is substantially lessthan the transverse cross sectional area of the chamber over asubstantial part of the height of the chamber above the orifice.

According to a further aspect of the present invention a method isprovided of operating a particle characterization apparatus comprising acartridge as disclosed herein, the cartridge being demountable from theapparatus, the method comprising sampling liquid containing particleswith the cartridge through the bore with the sampling member in itsfirst position, positioning the cartridge in the apparatus, moving thesampling member to its second position, pumping liquid in the storagechamber through the first cavity and into the first mixing chambertogether with the liquid sample, making particle characterizingmeasurements, disconnecting the cartridge from the apparatus, anddiscarding the cartridge.

Generally, in all embodiments it is preferred that all components, whichare wet by the sample in use, are disposable and all non-disposablecomponents can be re-used without cleaning.

It is an important advantage of the present invention that means forliquid sample preparation and analysis are integrated into a disposablecartridge. For example, the analytical steps comprise sampling of aprecise amount of blood, dilution of the amount of blood and finallymixing the blood with diluent into a homogeneous solution. The analysismay include spectrophotometric analysis of the liquid.

Thus, according to the present invention, means are provided forunambiguously making a blood analysis, such as counting the blood cellsin a small amount of blood coming from a droplet of capillary blood.Means are provided for taking an exact amount of blood sample, reagentspresent in the diluent may be added for e.g. dilution and/or chemicalpreparation of the sample, and the mixed sample and diluent flowsthrough a sensor for analysis of individual blood cells anddetermination of the volume of the analysed quantum of liquid.

As a supplement a spectrophotometric measurement can be performed inorder to quantify the content of e.g. haemoglobin.

The cartridge may comprise the following parts:

-   1. A liquid storage chamber-   2. A blood-sampling device-   3. A first mixing chamber-   4. A flow through sensor arrangement-   5. A first collection chamber-   6. A volume metering arrangement comprised of a chamber and two    connected flow channels-   7. A hydraulic connection for moving the liquid through the    cartridge

The concept of the disposable unit can be further combined with thefollowing additional parts:

-   A. Optical structures for optical liquid level measurement-   B. Electrodes for liquid level measurement-   C. Anti-coagulation treatment of surfaces-   D. Reagents in the diluent for modification of e.g. blood cells-   E. Mixing flee or baffle for assisted mixing-   F. Multiple volume metering arrangements for altering volumes-   G. A coating tape covering the sample inlet before use-   H. A waste chamber for waste/overflow-   I. A valve preventing liquid to exit through exhaust tube-   J. An integrated piston or membrane to replace an external source of    pressure-   K. A window for spectrophotometric measurements

The liquid storage chamber (part 1) holds the required amount of diluentused for the blood analysis. When the blood has been sampled into thecartridge, the diluent is flushed through the capillary to wash out thesampled blood and dilute it as required by the test. Dilutions of 100 to100.000 times are considered to be normal ratings and dilutions of 500to 10.000 times are preferred. The liquid storage chamber shouldpreferably be constructed to facilitate total draining of the chamber.This would be accomplished by having a slanting of the bottom of thechamber.

The sampling unit (part 2) may comprise a capillary extending through amovable rod placed in a tight-fitting supporting body. The movable rodis used for entrapment of a precise amount of blood sample. When bloodhas filled the capillary by capillary forces, the rod is turned and/ordisplaced from its initial position in the supporting body, thusisolating the part of the capillary that extends through the rod.

After moving the rod in the supporting body into its second position thecapillary forms a liquid path between the liquid storage chamber and thefirst mixing chamber (part 3). By applying a low pressure to the firstmixing chamber the diluent and blood sample is forced into the firstmixing chamber, where mixing will be performed by convection orsubsequently by blowing bubbles into the mixing chamber.

The flow through sensor arrangement (part 4) is comprised of a smallorifice in a membrane that establishes a liquid path from the firstmixing chamber to the first collection chamber. On each side of themembrane (in the first mixing chamber and in the first collectionchamber) an electrode is placed contacting the liquid.

The first collection chamber (part 5) forms a liquid priming function ofthe backside of the sensor system.

The volume metering system (part 6) is necessary for determination ofthe cell concentration. It comprises volume-metering chamber of a knownvolume with two relatively thin channels connecting the inlet at thebottom and the outlet at the top. Sensing of the liquid at the inlet andoutlet can be applied by optical or electrical means.

The outlet of the volume metering system is connected through a channel(part 7) to a source of pressure for moving the liquid through thecartridge.

The additional parts to the concept are further described here:

Addition A: Optical detection by change of optical properties of achannel such as changed reflectance or transmittance due to replacementof air with liquid in the channel. The surface over the inlet and outletof the volume-metering cell should be structured to optimize thecoupling of the light into the channel. The presence of liquid in atransparent polymer channel will result in a transmission of the signalsas opposed to a reflection when no liquid is present, which can beregistered by optical sensors.

Addition B: Two electrodes for liquid level measurement are connectedthrough the body of the cartridge into the inlet and outlet of thevolume-metering cell respectively. The electrodes will beshort-circuited through the saline liquid to the electrode placed in thefirst collection chamber, which can be registered through an externalelectrical arrangement.

Addition C: The anti-coagulation treatment of surfaces in the samplingstructure can be achieved by having selected compounds adhered orchemically bonded to these surfaces. Examples of such compounds areheparin and salts of EDTA.

Addition D: Reagent in the diluent for modification of e.g. blood cells.This reagent can consist of one or several compounds capable ofhemolysing the erythrocytes. In addition other compounds may be added inorder to: stabilize leukocytes and/or thrombocytes, adjust the pH-valueand osmotic pressure, minimize bacterial growth, modify the haemoglobinpresent and minimize batch to batch variations. The following exampleshave been included to provide information on relevant subjects relatedto the performance of a self-contained test cartridge.

Examples of compounds capable of selectively hemolysing the red bloodcells are: mixtures of quaternary ammonium salts as described in e.g.U.S. Pat. No. 4,485,175; U.S. Pat. No. 4,346,018; U.S. Pat. No.4,745,071; U.S. Pat. No. 4,528,274; and U.S. Pat. No. 5,834,315.

Examples of compounds capable of, during the hemolysis of the red bloodcells, stabilizing the leukocytes are N-(1-acetamido)iminodiacetic acid,procaine hydrochloride as described in e.g. U.S. Pat. Nos. 4,485,175 and1,3-dimethylurea as described in e.g. U.S. Pat. No. 4,745,071. Inaddition N-(1-acetamido)iminodiacetc acid is proposed to further assistthe quaternary ammonium salts in minimizing debris stemming fromhemolysed red blood cells as described in e.g. U.S. Pat. No. 4,962,038and adjust the pH-value (see below).

Examples of compounds added in order to adjust the pH-value and notleast importantly the osmotic pressure of the diluent are:N-(1-acetamido)iminodiacetic acid, sodium chloride, sodium sulphate asdescribed in e.g. U.S. Pat. No. 4,485,175 and U.S. Pat. No. 4,962,038.

Examples of compounds capable of minimizing bacterial growth are:1,3-dimethylolurea and chlorhexidine diacetate as described in e.g. U.S.Pat. No. 4,962,038.

Examples of compounds added to convert the hemoglobin species to anend-product suitable for spectrophotometric analysis are: potassiumcyanide as described in e.g. U.S. Pat. No. 4,485,175; U.S. Pat. No.4,745,071; U.S. Pat. No. 4,528,274 and tetrazole or triazole asdescribed in WO 99/49319.

Examples of particles or compounds which may be added in order tointroduce a tool for minimizing variation between different batches ofthe disposable device are: latex beads of known size and glass beads ofknown size.

Addition E: If assisted mixing is required the first mixing chambermight optionally include a mixing flee or a baffle. A magnetic flee maybe used to force the convection through an externally moving magneticfield. A baffle may be used to mechanically stir the liquid when movedby an externally connecting mechanical device. This could be required ifmixing with bubbles, such as bubbles blown into the sample through thesensor, is not adequate or possible.

Addition F: Multiple volume metering arrangements can be successivelyincluded if the test must deal with different concentrations of thedifferent particles.

Addition G: A lid or coating tape may be used to cover the sample inletbefore use. This ensures a clean sampling area at the origination of thetest.

Addition H: A waste chamber may be applied at the outlet of thevolume-metering cell for waste or overflow of liquid.

Addition I: At any connection ports, e.g. the connection port to thepressure source, a small valve can be integrated to prevent liquid toleak out of the cartridge.

Addition J: A piston or membrane can be integrated into the cartridge toinclude a source of pressure for moving the liquid. The piston ormembrane could be moved by a mechanical force provided by theinstrument.

Addition K: An optical window can be integrated into the cartridge inorder to perform optical measurements such as spectrophotometricdetection of the haemoglobin content in a blood sample.

The methods described can be combined to give the best solution for thefinal application. The disposable sensor is particularly usable whereportable, cheap, simple or flexible equipment is needed, such as insmall laboratories, in measurements in the field or as a “point of care”(“near-patient”) diagnostic tool.

When using the Coulter principle the diluent for use in the apparatusaccording to the invention may contain inorganic salts rendering theliquid a high electrical conductivity. When sample is applied to theelectrolyte, the electrolyte to sample volumes should preferably behigher than 10. Sample preparation should preferably result in between1.000 to 10.000.000 particles per ml and more preferably between 10.000and 100.000 particles per ml. A mixing of the sample after addingelectrolyte is recommended. Particle diameters should preferably bewithin 1 to 60 percent of the orifice diameter and more preferablybetween 5 to 25 percent of the orifice diameter. Volume flow shouldpreferably be from 10 μl to 10 ml per minute and more preferably between100 μl and 1 ml per minute. For the measurement a constant electricalcurrent of approximately 1 to 5 mA should preferably be applied. Thesource of electrical current should preferably have a signal to noiseratio (S/N) better than 1.000. The response from the electrodes can befiltered electronically by a band-pass filter.

According to yet another aspect of the invention a cartridge is providedcomprising a housing with a first mixing chamber and a first collectionchamber separated by a wall containing a first orifice for the passageof the particles between the first mixing chamber and the firstcollection chamber, first particle characterization means forcharacterizing particles passing through the first orifice, a bore inthe outer surface of the housing for entrance of the liquid sample,communicating with a first sampling member positioned in the housing forsampling the liquid sample and having a first cavity for receiving andholding the liquid sample, the member being movably positioned inrelation to the housing in such a way that, in a first position, thefirst cavity is in communication with the bore for entrance of theliquid sample into the first cavity, and, in a second position, thefirst cavity is in communication with the first mixing chamber fordischarge of the liquid sample into the first mixing chamber.

The cartridge may further comprise a second mixing chamber and a secondcollection chamber separated by a second wall containing a secondorifice for the passage of the particles between the second mixingchamber and the second collection chamber, second particlecharacterization means for characterizing particles passing through thesecond orifice.

In one embodiment of the invention, the first cavity is in communicationwith the first mixing chamber, when the first sampling member is in itsfirst position, for entrance of liquid from the first mixing chamberinto the first cavity, and, in a third position of the first samplingmember, the first cavity is in communication with the second mixingchamber for discharge of the liquid in the first cavity into the secondmixing chamber.

In another embodiment of the invention, the cartridge further comprisesa second sampling member positioned in the housing for sampling a smalland precise volume of liquid from the first mixing chamber and having asecond cavity for receiving and holding the sampled liquid, the memberbeing movably positioned in relation to the housing in such a way that,in a first position, the second cavity is in communication with thefirst mixing chamber for entrance of liquid from the first mixingchamber into the first cavity, and, in a second position, the secondcavity is in communication with the second mixing chamber for dischargeof the sampled liquid in the second cavity into the second mixingchamber.

The cartridge may further comprise a reagent chamber positioned adjacentto the first mixing chamber for holding a reagent to be entered into thefirst mixing chamber.

Preferably, the cartridge further comprises a breakable seal separatingthe reagent chamber from the first mixing chamber.

With this embodiment, different chemical treatment of different parts ofthe liquid sample may be performed.

Also with this embodiment, further dilution of the liquid sample may beperformed. The invention will be further described and illustrated withreference to the accompanying drawings in which:

FIG. 1 shows a cross sectional side view through the components of adisposable unit 85, referred to as the cartridge,

FIG. 2 shows the flow-through sensor concept FIG. 3 comprises anapparatus based on the disposable cartridge, a docking station 66 and areader 74,

FIG. 4 shows the cartridge with a build in piston,

FIG. 5 schematically illustrates the sampling procedure,

FIG. 6 is a plot of results obtained in Example 1,

FIG. 7 is a plot of results obtained in Example 2,

FIG. 8 is a plot of results obtained in Example 3,

FIG. 9 is a plot of results obtained in Example 4,

FIG. 10 is a plot of results obtained in Example 5,

FIG. 11 is a schematic illustration of the cartridge and hydraulicconnections in example 6,

FIG. 12 is a plot of the process described in example 7,

FIG. 13 is a plot of the process described in example 8,

FIG. 14 shows schematically a second embodiment of the cartridge,

FIG. 15 shows schematically a third embodiment of the cartridge, and

FIG. 16 shows in perspective an apparatus according to the invention.

FIG. 1

A disposable cartridge with a housing 85 for blood analysis comprises aliquid storage chamber 1 containing a liquid diluent 11, a firstsampling member 2 positioned in the housing 85 for sampling a bloodsample 8 and having a cavity 10 for receiving and holding the bloodsample 8, the member 2 being movably positioned in relation to thehousing 85 in such a way that, in a first position, the cavity 10 is incommunication with a bore 90 for entrance of the blood sample 8 into thecavity 10 by capillary forces, and, in a second position, the cavity 10is in communication with the liquid storage chamber 1 and a mixingchamber 3 for discharge of the blood sample 8 diluted by the liquiddiluent 11 into the mixing chamber 3. The mixing chamber 3 is separatedby a wall containing an orifice 59 from and a collection chamber 5 forthe passage of the blood sample 8 between the mixing chamber 3 and thecollection chamber 5. The wall containing the orifice 59 constitutes apart of a flow-through sensor 4.

A volume metering arrangement is connected to the collection chambercomprising a volume metering chamber 6 having the size of the volume tobe measured during the measurement with two connecting channels 12, 13of relatively diminutive internal volumes for registering liquid entryand exit by optical or electrical means, from the volume meteringchamber a channel 7 leads out to a connection port 67 where a pressurecan be applied.

FIG. 2

The flow-through sensor 4 has a dividing wall 91 with a relativelynarrow orifice 59 for the passage of particles suspended in liquid. Theorifice serves as a sensing zone for detection and measurement of theindividual cells. The orifice in the sensor may be formed as a countorifice for counting and sizing particles by an impedance method knownas Coulter counting. Particles can be aspirated through the orifice bypressure driven flow in either direction. When a saline or otherelectrolytic liquid solution is added to the chambers, the two chamberswill be electrically isolated from each other except for the route forcurrent flow provided by the passage through the orifice.

FIG. 3

The chambers on each side of the flow through sensor may have electrodes34, 35 extending from an external terminal 61, 62 through the base wall63 of the disposable unit and into a configuration facing the inside ofits respective chamber. The cartridge is placed in a docking station 66in a portable apparatus in order to carry out the test. The dockingstation 66 has a cup shaped housing having a base 70 and a circumambientsidewall 71. In the base 70 there are respective spring loadedelectrical connectors 64, 65 for contacting the terminals 61, 62 of thecartridge automatically when the cartridge is received as a push fitinto the docking station. There is also a conduit 68 passing through thebase wall 70 aligned with the conduit 67 of the cartridge. Conduit 67 atits opening into the upper face of the wall 70 has a seal 69, such ase.g. and O-ring for forming a gas tight connection with the lower faceof the base wall 63 of the cartridge. A vacuum pump 72 is connected by aline 73 to the lower end of the conduit 68. In a modification of theapparatus, the vacuum pump 72 can be reversed so as to apply positivegas pressure to the conduit 68. Schematically indicated at 74 are thefurther conventional components of a Coulter counter including all theelectronic circuitry and display equipment needed for the operation ofthe apparatus.

FIG. 4

As an alternative to the gas pump a piston 9 could be build into thecartridge for directly appliance of a negative or positive pressure.

FIG. 5

FIG. 5 schematically illustrates the blood sampling operation. Theillustrated part of the cartridge 2 includes the liquid storage chamber83 for storing a diluent for diluting the sample and the first mixingchamber 77 for mixing the sample 84 and the diluent. This figureschematically illustrates a device for sampling a small and accuratevolume of liquid in accordance with the present invention. The device 10comprises a first member 86 with a first opening 87 for entrance of aliquid sample into a bore 75 in the first member 86 and with a secondopening 76 for outputting the liquid sample from the bore 75. The bore75 forms a capillary tunnel. The first opening 87 of the first member 86may be brought into contact with a liquid 8 (shown in FIG. 1), 84 to besampled so that the liquid 84 may flow through the first opening 87 intothe bore 75 and out of the second opening 76 by capillary attraction.The device 12 further comprises a sampling member 78 with a first cavity82 for receiving and holding the liquid sample 84 and having a thirdopening 88 communicating with the first cavity 82. The first cavityforms a capillary tunnel with essentially the same diameter as the bore75. The sampling member 78 is a circular cylinder that is movablypositioned in relation to the first member 86. During sampling of theliquid, the sampling member 78 is positioned in the illustrated firstposition in relation to the first member 86 wherein the second opening76 is in communication with the third opening 88 so that sampled liquidmay flow through the second 76 and third opening 88 into the firstcavity 82 by capillary attraction. The third opening 88 may bedisconnected from the second opening 76 in a second position of thesampling member 78 in relation to the first member 86 so that the liquidsample 84 contained in the first cavity 82 is disconnected from the bore75.

The sampling member 78 is inserted into a third cavity 34 of the firstmember 86 for receiving and accommodating a part of the sampling member78. The sampling member 78 may be displaced between the first and secondposition along a longitudinal axis of the sampling member 78 that isalso substantially perpendicular to a longitudinal axis of the firstcavity 82. The sampling member 78 may also be rotatable about alongitudinal axis that is substantially perpendicular to a longitudinalaxis of the first cavity 82. In the first position, the first 75 andsecond 82 capillary tunnels extend along substantially the samelongitudinal center axis.

In the illustrated embodiment the first member 86 is symmetrical and hasa fourth cavity 80 with openings 81, 79 opposite the bore 75, and thesampling member 78 has an opening 89 opposite the opening 88 so that, inthe first position, a capillary tunnel extends through the first 86 andthe second 78 member and communicates with the environment throughopenings 87, 79. Thus, air may escape from the capillary tunnel throughopening 79. Further, in the first position, a part of the liquidentering the first cavity 82 will leave the cavity 82 through opening 89thereby ensuring that the cavity 82 has been completely filled withliquid during liquid sampling eliminating the risk of sampling with areduced sample volume leading to low accuracy sampling.

FIG. 5 a illustrates the device 2 ready for receiving the liquid. InFIG. 5 b, a sample has entered into the capillary tunnel 82, and in FIG.5 c the sampling member 78 has been rotated into the second position forisolation of an accurate volume of the sample 84, and finally FIG. 5 dillustrates that the sample 84 has been washed out of the capillarytunnel 82 and into the first mixing chamber 77 by the diluent.

Example: The capillary tunnel forming the first cavity 82 may have alength of 8 mm and a diameter of 0.9 mm for containing a liquid sampleof 5.089 μL.

Example: The capillary tunnel forming the first cavity 82 may have alength of 5 mm and a diameter of 0.5 mm for containing a liquid sampleof 0.982 μL.

Example: The capillary tunnel forming the first cavity 82 may have alength of 3 mm and a diameter of 0.3 mm for containing a liquid sampleof 0.212 μL.

FIG. 6

Example 1 Sizing of Polymer Beads

A mixture of 5 μm and 10 μm particles suspended in electrolyte wasaspirated through the orifice of the apparatus shown in FIG. 3. Thenumbers of particles detected and the size of each detected particlewere recorded. A bimodal distribution of detected particle size isclearly seen in FIG. 6.

FIG. 7

Example 2 Red Blood Cell Counting

Measurement of blood cells has been performed and the result is shown inFIG. 7. Red blood cells are normally around 5 to 7 μm in diameter andare the most frequent in whole blood, as can be seen on the FIG. 7. Thedistribution is a Gaussian curve, as it should be expected. Blood countscan be used in clinical diagnostics. It is fairly simple to counterythrocytes, leukocytes and thrombocytes by impedance measurements,which are considered the basic parameters for haematology (see“Fundamentals of Clinical Haematology”, Stevens, W.B. Saunders Company,ISBN 0-7216-4177-6).

FIG. 8

Example 3 White Cell Counting using a Diluent Containing aReagent-Composition Selected so as to Preserve all Blood Cells

Material

Cartridge and apparatus containing the functions as described in thepresent invention,

Isoton, Beckman Coulter (prod.no. 24655) containing: sodium chloride 7.9g/L, potassium chloride 0.4 g/L, disodiumhydrogenphosphate 1.9 g/l,sodiumdihydrogenphosphate 0.2 g/L, disodium-EDTA 0.4 g/L and sodiumfluoride 0.3 g/L.

Vacutainer, K3E, Becton & Dickinson, prod. No. 367652.

Bayer, ADVIA-120 equipment.

Performance

The full sequence of the procedure was as follows:

-   -   Collection of a venous blood sample in a vacutainer tube.    -   Leaving the sample, for the sedimentation process to proceed,        for three hours.    -   Extraction the plasma phase with the major part of the        buffy-coat section included    -   Performing analysis using the Bayer Advia 120 equipment for        obtaining a comparative value for the content of leukocytes.    -   Adding 5.00 ml isoton solution as diluent to the chamber of the        test rig    -   Adding 10.0 μl of sample to the chamber    -   Mixing liquids in the chamber    -   Starting test sequence on the computer (starts the pump and        readies the sampling)    -   When the liquid reaches the first level electrode sampling is        started    -   When the liquid reaches the second level electrode the sampling        is stopped    -   Sampled values are saved in a file    -   The file is opened with a “pulse-viewer” for data analyzing and        calculation of the result using a method of calculation        involving subtraction of, with the leukocytes overlapping red        blood cells.        Results

-   Bayer Advia-120: 11.96×10A9 leukocytes/L

-   Test-rig: 11.92×10ˆ9 leukocytes/L

-   Difference in accuracy: (11.96-1.92)/11.96=0.33%

FIG. 9

Example 4 White Cell Isolation using a Diluent Containing a ReagentComposition Selected so as to Primarily Hemolyse the Red Blood Cells

Material

Cartridge and apparatus containing the functions as described in thepresent invention,

Diluent containing: procaine hydrochloride 0.10 g/L, 1,3-dimethylolurea0.90 g/L, N-(1-acetamido)iminodiacetic acid 1.28 g/L, dodecyltrimethylammonium chloride 7.51 g/L and sodium chloride 0.03 g/L.

Vacutainer, K3EDTA, Becton & Dickinson, prod. No. 367652.

Performance

The full sequence of the procedure was as follows:

-   -   Collection of a venous blood sample in a vacutainer tube.    -   Leaving the sample, for the sedimentation process to proceed,        for three hours.    -   Extraction the plasma phase with the major part of the        buffy-coat section included    -   Adding 2.000 ml diluent as described above to the chamber of the        test rig    -   Adding 4.0 μl of sample to the chamber    -   Mixing liquids in the chamber    -   Starting test sequence on the computer (starts the pump and        readies the sampling)    -   When the liquid reaches the first level electrode sampling is        started    -   When the liquid reaches the second level electrode the sampling        is stopped    -   Sampled values are saved in a file    -   The file is opened with a “pulse-viewer” for data analyzing and        generation of the result.        Results

As can be seen in the histogram in FIG. 6 the particle populationcorresponding to the leukocytes is easily identified in the absence ofthe red blood cells.

FIG. 10

Example 5 Counting Somatic Cells

Milk quality is essential for farmers, diary producers and consumers.Farmer has to deliver milk of a certain quality, which is controlled bythe so-called Somatic Cell Count (SCC). In milk quality tests somaticcells in the milk are counted to determine infections (clinicalmastitis). A limit of 400.000 cells pr. ml. has to be met by the farmersfor dairy resale. Change of diet, stress or mastitis lead to higher SCClevels, thus lowering the quality of the milk and consequently loweringthe price per unit volume. A cheap cell counter will help farmers anddiary producers monitor SCC-level.

FIG. 11

Example 6 A Blood Diagnostic System

This is an example of a 3 part differential white blood cell count(monocytes, lymphocytes, granulocytes), thrombocytes count andhaemoglobin measurement and the corresponding instrumentation andcartridge realized through the present invention.

A three-part differentiation of white blood cells, thrombocyte counterwith measurement of haemoglobin can be achieved with the specifiedcomponents.

A reagent for selectively lysing red blood cells is added to the diluentin the storage chamber 1. When the whole blood 8 is added to the opening58 of the first capillary section 15, the blood will be dragged in tothe capillary and through the middle section 10 and last section 14 ofthe capillary. The last section of the capillary is connected to afill-chamber 43 for visually verification of the filling. Thefill-chamber 43 is connected through a conduct 44 to open air.

The blood filled middle section of the capillary is part of a knob 2that can be moved to a second position, connecting the ends of thecapillary to two other conducts, a conduct 45 connected to the storagechamber 1 and a second conduct 40 connected to the first mixing chamber3 respectively. A third conduct 39 is leading from the first mixingchamber to a port opening 42 in the cartridge. The port opening isconnected through a counter port opening 37 in the apparatus, through atubing 46 to a three-position valve 51 and directed through the twopositions of the valve to open air through a second tubing 55 or througha third tubing 50 to the suction port of a membrane pump 47.

When the blood and diluent with reagent has been sucked into the firstmixing chamber, the blood can be mixed by blowing bubbles through theorifice of the sensor 4. The air pressure is applied through thecollection chamber 5, via a fourth conduct 12A, a small volume chamber6A, a fifth conduct 12B, a large volume chamber 6B and a sixth conduct 7directed to an opening port 41 in the cartridge. A counter port 36 inthe apparatus is connected through a fourth tubing 48 to a second threeposition valve 52, which has positions to direct to both vacuum througha fifth tubing 56 to the suction port of the membrane pump, or to theexhaust of the membrane pump, through a third two position valve 53 anda sixth tubing 49, the third valve having two positions for theconnection and for directing the pump exhaust to open air through aseventh tubing 54 respectively.

After mixing the diluted and lysed blood (red blood cells is removed) itis ready to be measured. The first mixing chamber is connected throughthe first valve to open air and the collection chamber is connectedthrough the second valve to the suction port of the pump. The exhaust ofthe membrane pump is connected through the third valve to open air. Asthe blood and diluent flows from the first mixing chamber into thecollection chamber, an electrical connection between to counterelectrodes 34 and 35 placed in each chamber is established through theliquid. Cells are counted and differentiated by size by the Coulterprinciple. Through sizing of the cells, the cells can be distinguishedand categorised into different groups containing cells of a certaintype. Thus white blood cells (leucocytes) can be differentiated intogranulocytes, lymphocytes and monocytes. Furthermore, thrombocytes(platelets) can be differentiated from leucocytes as well. In order todetermine the concentration, the volume of the diluted blood, which hasbeen counted, must be known. Since thrombocytes are approximately tentimes as frequent as leucocytes, it may be necessary to measure twodifferent volumes. The thrombocytes are counted according to a smallvolume chamber 6A positioned between the collection chamber and thelarger volume. By registering the liquid entry and exit at the inlet andoutlet of the small volume chamber respectively, the counting periodwill be given. Registration of the liquid level is preferably done by anoptical reflectance measurement at the inlet 33 and at the outlet 32.The outlet of the small volume chamber is also the inlet of the largevolume chamber 6B. This chamber is used in connection with counting ofleucocytes. At the outlet of the large volume chamber, a third opticalreflectance measurement 31 is performed to register the exit of theliquid from this chamber.

After counting both leucocytes and thrombocytes the haemoglobin contentcan be measured by optical spectroscopy preferably through the middlesection of the large volume chamber 30.

Process of the test (example 6):

The process of making a test by means of the present invention can becharacterized as:

-   -   1) Draw blood by using a lancet device    -   2) Pick up blood droplet by touching the blood to the cartridge        inlet    -   3) Mount cartridge in the instrument (instrument starts and runs        the test)    -   4) Read the result from the display    -   5) Remove and discard cartridge FIG. 12

Example 7 Photolithography

An orifice may suitably be formed in a photo-reactive polymer byphotolithography and subsequent development. Thus a free standing sheetof polymer of the kind used conventionally as a photo resist materialmay be exposed to light to render a spot to soluble to define an orifice(or to render the non-spot forming areas in-soluble) followed bydevelopment with solvent to remove material to form the orifice.Normally, a large number of count wafers each containing a respectiveorifice will be made simultaneously in one sheet. Suitable photo resistpolymers are described in e.g. M. Madou “Fundamentals of Microfabrication, CRC Press LLC, 1997, ISBN 0-8493-9451-1. They includeAZ-5214E, SU8, polyamides and others.

Alternatively, the photo resist polymer may be used as a protectinglayer over a substrate such as silicon in which the orifice is formed byetching regions exposed by development of the photo resist. If theetched substrate is electrically conducting it may be insulated prior touse by the formation of a suitable insulating layer there over. Thephoto resist polymer may be used as such a layer.

Count wafers made lithographically may be used in all forms of apparatusand method according to this invention. FIG. 12 shows one process offabricating the count wafer: (a) appliance of a thin sheet of photoresist. (b) Development of the mask. (c) Etching of the orifice by DeepReactive Ion Etching (DRIE, M. Madou “Fundamentals of Micro fabrication,CRC Press LLC, 1997, ISBN 0-8493-9451-1).

FIG. 13

Example 8 Orifice Fabricated by Laser Micro Machining

Orifices for Coulter counting can be fabricated by laser micro machiningof polymers, which could lead to a simple and convenient way offabricating and assembling orifices for the cartridge. A series of smallholes of 50 μm has been fabricated with an UV-laser. The holes are madein less than 1 ms in a 50 μm polymer sheet. The uniformity of the holesis very high and the smoothness of the orifice entrance is unique. FIG.13 shows the process of laser machining of the orifice. The laser cutsthrough the polymer foil in a circle, thus defining the size of theorifice.

FIG. 14

FIG. 14 shows schematically a preferred embodiment of the cartridgeaccording to the invention. The illustrated cartridge has a first member104 for sampling blood. The member 104 is movably positioned in relationto the housing 100 between three positions, a first position for bloodsampling, a second position to connect the first storage chamber 103with the first mixing chamber 112, and a third position to connect thesecond storage chamber 105 with the second mixing chamber 110. The bloodis passed through the bore 122 into the first cavity of the member 104by capillary forces or by applying a vacuum at the end of the samplingchannel 111. A liquid blocking valve 116 is arranged after the firstsampling member to hinder passage of blood through the channel. Afterthe blood sampling, the sampling member is turned to the second positionand the sample is flushed into the first mixing chamber 112 by theliquid in the first storage chamber 103. In the first mixing chamber 112the sample is diluted 1:200 with the liquid in the first storage chamber103 and a fraction is blown back into the first cavity of the samplingmember 104, which is turned to the third position so that the dilutedsample is flushed into the second mixing chamber 110 by the liquid inthe second storage chamber 105. In the second mixing chamber 110 thesample is further diluted 1:200 to a total dilution of 1:40.000 with theliquid in the second storage chamber 105. A hemolysing reagent isinjected into the first mixing chamber 112 by a piston 115, which breaksa seal 118 between a reagent chamber 119 and the first mixing chamber112. After hemolysing the blood the 1:200 diluted sample is ready forcounting non-hemolysed white blood cells and for measuring hemoglobin byphotometry. The white cells are counted by passing them through a firstorifice 113 and measuring the response by impedance cell counting over afirst electrode pair 117, 120. A fixed volume is counted by a firstvolume metering arrangement 107 connected to the first collectionchamber 114. A first overflow volume 106 is arranged after the firstvolume metering arrangement 107. The white blood cells can bedifferentiated by volume after adding the lysing reagent to the blood.The white cells can be grouped by volume into: Granulocytes, Monocytesand Lymphocytes. The three groups together yield the total white cellcount.

In the second mixing chamber 110, red cells and platelets are counted.The red cells and platelets are counted by passing them through a secondorifice 109 and measuring the response by impedance cell counting over asecond electrode pair 106, 121. A fixed volume is counted by a secondvolume metering arrangement 101 connected to the second collectionchamber 108. A second overflow volume 102 is placed after the secondvolume metering arrangement 101.

The embodiment may further comprise an additional optical detector forphotometric determination of the hemoglobin content. Referred to simplyas “total hemoglobin”, this test involves lysing the erythrocytes, thusproducing an evenly distributed solution of hemoglobin in the sample.The hemoglobin is chemically converted to the more stable and easilymeasured methemoglobintriazole-complex, which is a colored compound thatcan be measured calorimetrically, its concentration being calculatedfrom its amount of light absorption using Beer's Law. The methodrequires measurement of hemoglobin at approx. 540 nm where theabsorption is high with a turbidity correction measurement at 880 nmwhere the absorption is low.

FIG. 15

FIG. 15 shows schematically another preferred embodiment of thecartridge according to the invention. The illustrated cartridge has afirst member 104 for sampling blood. The member 104 is movablypositioned in relation to the housing 100 between two positions, a firstposition for blood sampling, and a second position to connect the firststorage chamber 103 with the first mixing chamber 112. A blood sample ispassed through the bore 122 into the first cavity of the member 104 bycapillary forces or by applying a vacuum at the end of the samplingchannel 111. A liquid blocking valve 116 is arranged after the firstsampling member to hinder passage of blood through the channel. Afterthe blood sampling, the sampling member is turned to the second positionand the sample is flushed into the first mixing chamber 112 by theliquid in the first storage chamber 103. In the first mixing chamber 112the sample is diluted 1:200 with the liquid in the first storage chamber103.

The cartridge further comprises a second sampling member 123 positionedin the housing 100 for sampling a small and precise volume of liquidfrom the first mixing chamber 112 and having a second cavity 123 forreceiving and holding the sampled liquid, the member 123 being movablypositioned in relation to the housing 100 in such a way that, in a firstposition, the second cavity 123 is in communication with the firstmixing chamber 112 for entrance of a diluted sample from the firstmixing chamber 112 into the second cavity 123, and, in a secondposition, the second cavity 123 is in communication with the secondmixing chamber 110 so that the diluted sample is flushed into the secondmixing chamber 110 by the liquid in the second storage chamber 105. Inthe second mixing chamber 110 the sample is further diluted 1:200 to atotal dilution of 1:40.000 with the liquid in the second storage chamber105. A hemolysing reagent is injected into the first mixing chamber 112by a piston 115, which breaks a seal 118 between a reagent chamber 119and the first mixing chamber 112. After hemolysing the blood the 1:200diluted sample is ready for counting non-hemolysed white blood cells andfor measuring hemoglobin by photometry. The white cells are counted bypassing them through a first orifice 113 and measuring the response byimpedance cell counting over a first electrode pair 117, 120. A fixedvolume is counted by a first volume metering arrangement 107 connectedto the first collection chamber 114. A first overflow volume 106 isarranged after the first volume metering arrangement 107. The whiteblood cells can be differentiated by volume after adding the lysingreagent to the blood. The white cells can be grouped by volume into:Granulocytes, Monocytes and Lymphocytes. The three groups together yieldthe total white cell count.

In the second mixing chamber 110, red cells and platelets are counted.The red cells and platelets are counted by passing them through a secondorifice 109 and measuring the response by impedance cell counting over asecond electrode pair 106, 121. A fixed volume is counted by a secondvolume metering arrangement 101 connected to the second collectionchamber 108. A second overflow volume 102 is placed after the secondvolume metering arrangement 101.

The embodiment may further comprise an additional optical detector forphotometric determination of the hemoglobin content. Referred to simplyas “total hemoglobin”, this test involves lysing the erythrocytes, thusproducing an evenly distributed solution of hemoglobin in the sample.The hemoglobin is chemically converted to the more stable and easilymeasured methemoglobintriazole-complex, which is a colored compound thatcan be measured calorimetrically, its concentration being calculatedfrom its amount of light absorption using Beer's Law. The methodrequires measurement of hemoglobin at approx. 540 nm where theabsorption is high with a turbidity correction measurement at 880 nmwhere the absorption is low.

1-28. (canceled)
 29. A cartridge for characterizing particles suspendedin a liquid sample, comprising a housing with connectors for operationalconnection to and disconnection from corresponding connectors of adocking station for establishment of electrical and fluid connectionswhen the cartridge is received in the docking station, a first mixingchamber and a first collection chamber separated by a wall containing afirst orifice for the passage of the particles between the first mixingchamber and the first collection chamber, first particlecharacterization means for characterizing particles passing through thefirst orifice, a bore in the outer surface of the housing for entranceof the liquid sample, communicating with a first sampling memberpositioned in the housing for sampling the liquid sample and having afirst cavity for receiving and holding the liquid sample, the memberbeing movably positioned in relation to the housing in such a way that,in a first position, the first cavity is in communication with the borefor entrance of the liquid sample into the first cavity, and, in asecond position, the first cavity is in communication with the firstmixing chamber for discharge of the liquid sample into the first mixingchamber whereby the sampling member operates to receive and hold aprecise volume of liquid sample and to transfer the sample to the firstmixing chamber.
 30. A cartridge according to claim 29, furthercomprising a second mixing chamber and a second collection chamberseparated by a second wall containing a second orifice for the passageof the particles between the second mixing chamber and the secondcollection chamber, second particle characterization means forcharacterizing particles passing through the second orifice, and whereinin the second position, the first cavity is in communication with thefirst mixing chamber for entrance of liquid from the first mixingchamber into the first cavity, and, in a third position, the firstcavity is in communication with the second mixing chamber for dischargeof the liquid in the first cavity into the second mixing chamber.
 31. Acartridge according to claim 29, further comprising a second mixingchamber and a second collection chamber separated by a second wallcontaining a second orifice for the passage of the particles between thesecond mixing chamber and the second collection chamber, second particlecharacterization means for characterizing particles passing through thesecond orifice, and a second sampling member positioned in the housingfor sampling a small and precise volume of liquid from the first mixingchamber and having a second cavity for receiving and holding the sampledliquid, the member being movably positioned in relation to the housingin such a way that, in a first position, the second cavity is incommunication with the first mixing chamber for entrance of liquid fromthe first mixing chamber into the first cavity, and, in a secondposition, the second cavity is in communication with the second mixingchamber for discharge of the sampled liquid in the second cavity intothe second mixing chamber.
 32. A cartridge according to claim 29,further comprising a reagent chamber positioned adjacent to the firstmixing chamber for holding a reagent to be entered into the first mixingchamber.
 33. A cartridge according to claim 32, further comprising abreakable seal separating the reagent chamber from the first mixingchamber.
 34. A cartridge according to claim 29, wherein at least one ofthe first and second particle characterization means includes a firstelectrode in the respective one of the first and second mixing chamberand a second electrode in the respective one of the first and secondcollection chamber, each electrode being electrically connected to arespective terminal member accessible at the outer surface of thecartridge.
 35. A cartridge according to claim 29, wherein the housingfurther comprises a first liquid storage chamber for holding a liquidand that, in the second position of the first sampling member,communicates with the first cavity so that liquid can be discharged fromthe first liquid storage chamber through the first cavity of the firstsampling member and into the first mixing chamber together with theliquid sample.
 36. A cartridge according to claim 29, wherein thehousing further comprises a second liquid storage chamber for holding aliquid to be discharged from the second liquid storage chamber throughthe respective one of the first and second cavity and into the secondmixing chamber together with the sampled liquid.
 37. A cartridgeaccording to claim 29, comprising volume metering means for determiningthe beginning and end of a period during which a predetermined volume ofliquid has passed through at least one of the first and second orifice.38. A cartridge according to claim 37, wherein the volume metering meanscomprises a volume metering chamber with an input communicating with therespective collection chamber and an output, and wherein presence ofliquid is detected at the input and at the output, respectively.
 39. Acartridge according to claim 38, wherein presence of liquid is detectedwith a secondary electrode positioned at the input and a furthersecondary electrode positioned at the output.
 40. A cartridge accordingto claim 38, wherein presence of liquid is detected optically.
 41. Acartridge according to claim 29, wherein each of the mixing chambers andthe collection chambers has a transverse cross-sectional area at thelevel of the respective orifice which is substantially less than thetransverse cross-sectional area of the respective chamber over asubstantial part of the height of the chamber above the respectiveorifice.
 42. A cartridge according to claim 29, wherein the surfacedefining the first cavity of the first sampling member has ananti-coagulation reagent.
 43. A cartridge according to claim 29, whereinthe first liquid storage chamber holds chemical reagents formodification of the blood sample.
 44. A cartridge according to claim 29,wherein a mixing member is positioned in at least one of the mixingchambers.
 45. A cartridge according to claim 44, wherein the mixingmember is magnetic.
 46. A cartridge according to claim 29, furthercomprising a sensor for characterization of the liquid.
 47. A cartridgeaccording to claim 46, wherein the sensor for characterization of theliquid is adapted for spectrophotometric characterization of the liquid.48. A cartridge according to claim 29, wherein the housing furthercomprises a pump chamber communicating with one of the first and secondcollection chambers and having a pump actuator for causing a liquid flowthrough the respective orifice.
 49. A cartridge according to claim 48,wherein the pump actuator is a piston.
 50. A cartridge according toclaim 48, wherein the pump actuator is a membrane.
 51. A method ofoperating a particle characterization apparatus comprising a cartridgeaccording to claim 29, the cartridge being demountable from theapparatus, the method comprising sampling liquid containing particleswith the cartridge through the bore with the first sampling member inits first position, positioning the cartridge in the apparatus, movingthe first sampling member to its second position, pumping liquid in thefirst storage chamber through the second cavity and into the firstmixing chamber together with the liquid sample, making particlecharacterizing measurements, disconnecting the cartridge from theapparatus, and discarding the cartridge.
 52. A method of operating aparticle characterization apparatus comprising a cartridge according toclaim 31, the cartridge being demountable from the apparatus, the methodcomprising sampling liquid containing particles with the cartridgethrough the bore with the first sampling member in its first position,positioning the cartridge in the apparatus, moving the first samplingmember to its second position, pumping liquid in the first storagechamber through the first cavity and into the first mixing chambertogether with the liquid sample, sampling a liquid sample from the firstmixing chamber with the second sampling member in its first position,moving the second sampling member to its second position, pumping liquidin the second storage chamber through the second cavity and into thesecond mixing chamber together with the liquid sample, making particlecharacterizing measurements, disconnecting the cartridge from theapparatus, and discarding the cartridge.
 53. An apparatus forcharacterizing particles suspended in a liquid, comprising a cartridgeaccording to claim 29, and a docking station for removably receiving thecartridge, comprising connectors for operational connection with theparticle characterization means when the cartridge is received in thedocking station.
 54. An apparatus according to claim 53, wherein thecartridge further comprises a first port communicating with the firstcollection chamber for causing a liquid flow through the first orifice,and the docking station further comprises a port for forming a gasconnection with the with the cartridge port when the cartridge isreceived in the docking station for application of a pressure causing aliquid flow through the orifice.
 55. An apparatus according to claim 53,comprising a cartridge according to claim 53, the docking stationfurther comprising connectors for operational connection with the secondparticle characterization means when the cartridge is received in thedocking station.
 56. An apparatus according to claim 55, wherein thecartridge further comprises a second port communicating with the secondcollection chamber for causing a liquid flow through the second orifice,and the docking station further comprises a second port for forming agas connection with the with the second cartridge port when thecartridge is received in the docking station for application of apressure causing a liquid flow through the second orifice.